The diterpenoid anti-cancer drug paclitaxel and related taxanes accumulate in resin cells of Taxus (yew) stem, needle and root tissue (Croom, E. M., Jr. (1995) in Taxol: Science and Applications (Suffness, M., Ed.), pp. 37-70, CRC Press, Boca Raton, Fla.), and in cell cultures of yew (Ketchum, R. E. B. and Gibson, D. M. (1996) Plant Cell Tiss. Org. Cult., 46:9-16; Christen, A. A. et al (1989) Proc. Am. Assoc. Cancer Res., 30:566 [See also U.S. Pat. No. 5,019,504]) in which production is induced by methyl jasmonate (Yukimune, Y., Tabata, H., Higashi, M. and Hara, Y. (1996) Nature Biotech., 14:1129-1132; Ketchum, R. E. B., Gibson, D. M., Croteau, R. B. and Shuler, M. L. (1998) Biotech. Bioeng., in press). Paclitaxel has been approved for treatment of refractory ovarian and metastatic breast cancer and, more recently, for small cell lung cancer (Rose, W. C. (1995) in Taxol: Science and Applications (Suffness, M., Ed.), pp. 209-236, CRC Press, Boca Raton, Fla.), and ongoing clinical trials suggest expanded applications in cancer chemotherapy, both in treatment of additional cancer types and in use much earlier in the course of intervention (Holmes, F. A., Kudelka, A. P., Kavanagh, J. J., Huber, M. H., Ajani, J. A. and Valero, V. (1995) in Taxane Anticancer Agents: Basic Science and Current Status (Georg, G. I., Chen, T. T., Ojima, I. and Vyas, D. M., Eds.), pp. 31-57, American Chemical Society, Washington, D.C.). The supply and cost of the drug therefore remain important issues (Suffness, M. (1995) in Taxane Anticancer Agents: Basic Science and Current Status (Georg, G. I., Chen, T. T., Ojima, I. and Vyas, D. M., Eds.), pp. 1-17, American Chemical Society, Washington, D.C.).
Paclitaxel has been prepared by total synthesis (Nicolaou, K. C., Yang, Z., Liu, J. J., Ueno, H., Nantermet, P. G., Guy, R. K., Claiborne, C. F., Renaud, J., Couladouros, E. A., Paulvannin, K. and Sorensen, E. J. (1994) Nature, 367:630-634; Holton, R. A., Kim, H. B., Somoza, C., Liang, F., Biediger, R. J., Boatman, P. D., Shindo, M., Smith, C. C., Kim, S., Nadizadeh, H., Suzuki, Y., Tao, C., Vu, P., Tang, S., Zhang, P., Murthi, K. K., Gentile, L. N. and Liu, J. H. (1994) J. Am. Chem. Soc., 116:1599-1600; Masters, J. J., Link, J. T., Snyder, L. B., Young, W. B. and Danishefsky, S. J. (1995) Angew. Chem. Int. Ed. Engl., 34:1723-1726.); however, the synthetic routes are long, expensive, and too low yielding to be commercially useful (Borman, S. (1994) Chem. Eng. News, 72:32-34), and it is clear that, for the foreseeable future, the supply of this drug must continue to rely on biological methods of production (Suffness, M. (1995) in Taxane Anticancer Agents: Basic Science and Current Status (Georg, G. I., Chen, T. T., Ojima, I. and Vyas, D. M., Eds.), pp. 1-17, American Chemical Society, Washington, D.C.). It is therefore essential to understand the biosynthesis of paclitaxel, particularly the rate-limiting steps of the pathway, since the manipulation of these slow steps can be expected to lead to improved yield and to the production of the drug in large quantities at reasonable cost.
Paclitaxel is formed by the cyclization of the universal diterpenoid precursor geranylgeranyl diphosphate (West, C. A., Dudley, M. W. and Dueber, M. T. (1979) Recent Adv. Phytochem., 13:163-198; West, C. A. (1981) in Biosynthesis of Isoprenoid Compounds (Porter, J. W. and Spurgeon, S. L., Eds.), Vol. 1, pp. 375-411, Wiley, New York, N.Y.; Gershenzon, J. and Croteau, R. (1993) in Lipid Metabolism in Plants (Moore, T. S., Jr., Ed.), pp. 339-388, CRC Press, Boca Raton, Fla.) to taxa-4(5),11(12)-diene (Koepp, A. E., Hezari, M., Zajicek, J., Stofer Vogel, B., LaFever, R. E., Lewis, N. G. and Croteau, R. (1995) J. Biol. Chem., 270:8686-8690) to establish the taxane skeleton, which then undergoes extensive oxidative modification and addition of side chains (Hezari, M. and Croteau, R. (1997) Planta Med., 63:291-295) (FIG. 1). The properties and mechanism of taxadiene synthase have been examined in some detail (Hezari, M., Lewis, N. G. and Croteau, R. (1995) Arch. Biochem. Biophys., 322:437-444; Lin, X., Hezari, M., Koepp, A. E., Floss, H. G. and Croteau, R. (1996) Biochemistry, 35:2968-2977), the corresponding cDNA has been cloned (Wildung, M. R. and Croteau, R. (1996) J. Biol. Chem., 271:9201-9204), and several of the subsequent cytochrome P450-catalyzed hydroxylations (Hezari, M. and Croteau, R. (1997) Planta Med., 63:291-295; Hefner, J., Rubenstein, S. M., Ketchum, R. E. B., Gibson, D. M., Williams, R. M. and Croteau, R. (1996) Chem. Biol., 3:479-489) and acylation steps (Zocher, R., Weckwerth, W., Hacker, C., Kammer, B., Hornbogen, T. and Ewald, D. (1996) Biochem. Biophys. Res. Commun., 229:16-20) of the pathway have been demonstrated.
Genes encoding GGPP synthase are of interest because this branch point prenyltransferase (West, C. A., Dudley, M. W. and Dueber, M. T. (1979) Recent Adv. Phytochem., 13:163-198; West, C. A. (1981) in Biosynthesis of Isoprenoid Compounds (Porter, J. W. and Spurgeon, S. L., Eds.), Vol. 1, pp. 375-411, Wiley, New York, N.Y.; Gershenzon, J. and Croteau, R. (1993) in Lipid Metabolism in Plants (Moore, T. S., Jr., Ed.), pp. 339-388, CRC Press, Boca Raton, Fla.) provides the substrate for protein prenylation (Rilling, H. C., Breunger, E., Epstein, W. W. and Crain, P. F. (1989) Science, 247:318-320; Clarke, S. (1992) Annu. Rev. Biochem., 61:355-386.), the formation of the phytol moiety of chlorophylls (Kleinig, H. (1989) Annu. Rev. Plant Physiol. Plant Mol. Biol., 40:39-59), side-chain syntheses of prenylated quinones and tocopherols (Schultz, G., Soll, J., Fiedler, E. and Schulze-Siebert, D. (1985) Physiol. Plant., 64:123-129), the production of carotenoid pigments (Chappell, J. (1995) Plant Physiol., 107:1-6; Bonk, M., Hoffman, B., Von Lintig, J., Schledz, M., Al-Babili, S., Hobeika, E., Kleinig, H. and Beyer, P. (1997) Eur. J. Biochem., 247:942-950; Bartley, G. E. and Scolnik, P. A. (1994) Annu. Rev. Plant Physiol. Plant Mol. Biol., 45:287-301; Bartley, G. E. and Scolnik, P. A. (1995) Plant Cell, 7:1027-1038.) and gibberellin plant hormones (Sun, T. and Kamiya, Y. (1994) Plant Cell, 6:1509-1518), as well as for the biosynthesis of diterpenoid natural products, such as casbene (Dudley, M. W., Green, T. R. and West, C. A. (1986) Plant Physiol., 81:343-348), oryzalexins (West, C. A., Lois, A. F., Wickham, K. A. and Ren, Y. -Y. (1990) Recent Adv. Phytochem., 24:219-248) and paclitaxel (Koepp, A. E., Hezari, M., Zajicek, J., Stofer Vogel, B., LaFever, R. E., Lewis, N. G. and Croteau, R. (1995) J. Biol. Chem., 270:8686-8690). Since GGPP synthase controls the rate of production of the branch point precursor of paclitaxel, and other useful diterpenes, there exists a need for methods of enhancing the production of GGPP synthase in cells and tissues that produce paclitaxel and other useful diterpenes.